Molecules are composed of atoms bonded together. This bonding process is accomplished by the sharing of electrons. When two atoms come together and their electrons pair up, a bond is created. Generally, only two electrons can exist in one bond. Paired electrons are quite stable and almost all electrons in the human body exist in a paired state. However, when a bond is broken, the electrons can either stay together or split up. If the electrons stay together, both electrons go to one of the atoms and none go to the other atom. In this case the molecular fragments are called ions, which are electrically charged and typically not harmful to humans or other animals. For example, sodium chloride, NaCl, can split up into a sodium cation (Na+) and a chloride anion (Cl−). But, if the electrons split up when a bond is broken, one electron will go to each atom, creating two molecules with unpaired electrons, called free radicals. The unpaired electrons of these free radicals are highly energetic and unstable and seek out other electrons with which to pair with. As a result, free radicals steal electrons from other molecules. The process of stealing electrons from other electron pairs is what makes free radicals dangerous because it causes oxidation, or loss of electrons, to the molecule it attacks, leaving an unstable, highly energetic molecule. Since most electrons exist in a paired state, free radicals often end up reacting with paired electrons and create still more free radicals. Only when a free radical pairs up with another free radical is the free radical terminated.
Antioxidants, or free radical scavengers, function by offering easy electron targets for free radicals. In absorbing a free radical, antioxidants “trap”, or de-energize and stabilize the lone free-radical electron and make it stable enough not be harmful.
As such, antioxidants provide a defense against free radicals which cause cell oxidation in humans and other animals. Presently, there is overwhelming evidence to indicate that free radicals cause oxidative damage to lipids, proteins and nucleic acids. Antioxidants can play an important role in the prevention of a number of diseases including cancer, heart, vascular, and neurogenitive diseases. See Oxygen-Radical Absorbance Capacity for Antioxidants, Cao, G., Free Radical Biol. Med. Vol. 14, (1993), incorporated herein in its entirety by this reference.
Many foods contain substantial quantities of antioxidants. The need to effectively measure the antioxidant capacity of such foods is of significant importance to people who are trying to prevent diseases caused by free radicals, to manufacturers of foods alleged to contain high antioxidant capacities, and to the scientific community. Moreover, in the medical community, measuring the antioxidant capacity of blood and serum can be useful in prevention of disease. Accordingly, many food, vitamin and supplement suppliers seek to test the antioxidant capacity of their various products. In addition, biological samples are often tested to determine their antioxidant capacity.
In 1993, the Oxygen Radical Absorbance Capacity (ORAC) assay was developed to test the antioxidant capacity of a given sample. See Oxygen-Radical Absorbance Capacity for Antioxidants cited above. And, in 1998, an automated device, the Roche COBAS FARA II analyzer, was placed on the market to test samples according to the ORAC assay. Moreover, significant research has been performed to determine the antioxidant capacity of samples using the ORAC assay. See, e.g. Oxygen Radical Absorbance Capacity (ORAC) and Phenolic and Anthocyanin Concentrations in Fruit and Leaf Tissues of Highbush Blueberry, Ehlenfeldt, M. and Prior, R., J. Agric. Food Chem., 49, pp. 2222–2227 (2001); In Vivo Total Antioxidant Capacity: Comparison of Different Analytical Methods, Prior, R. and Cao, G., Free Radical Biol. Med., Vol. 27, Nos. 11/12, pp. 1173–1181 (1999); Total Antioxidant Capacity of Fruits, Wang, H., Cao, G., Prior, R., J. Agric. Food Chem., 44, pp. 701–705 (1996); and Antioxidant Capacity of Tea and Common Vegetables, Cao, G., Sofic, E., and Prior, R., J. Agric. Food Chem., 44, pp. 3426–3431 (1996), all incorporated herein in their entirety by this reference.
Since about 1998, the inventors hereof have used the COBAS FARA II to test various samples according to the ORAC assay. In performing the ORAC assay numerous times, the inventors hereof detected and herein delineate solutions to numerous problems associated with the conventional ORAC assay.
In accordance with the published ORAC assay, a sample such as fresh fruit, blood senun, or an additive or supplement in powder form is prepared for extraction and extracted first in water and then in acetone. A protein based fluorescent probe, namely B-phycoerythrin (B-PE) is then added to the extract. A standard, having high antioxidant capacity, such as diluted grape seed extract (GSE) or TROLOX® (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water soluble analog of vitamin E, is added to the extract to provide a comparison of the antioxidant capacity of the sample to the standard. The extract, the standard, and a blank sample are then loaded into the COBAS FARA II device and an initial fluorescence emission of the probe is taken. Next, AAPH, (2,2′-azobis (2-amidino-propane) dihydrochloride), which generates free radicals upon heating, is added to the extract of the sample and the standard and fluorescence emission readings are taken until a zero value is reached for the extract of the sample. To measure the protective effect of an antioxidant using the ORAC assay, the area under the fluorescence decay curve (AUC) of the sample is calculated and compared to that of the blank in which no antioxidant is present.
One problem with this prior art assay is that samples including high levels of lipid soluble antioxidants are not correctly rated because of their insolubility in aqueous media. Further, samples including both lipid soluble antioxidants and water-soluble antioxidants are not correctly rated.
Another problem with the prior art ORAC assay is that the probe used was B-PE. This protein probe was found to interact with the sample in adverse ways and generated false low readings. Moreover, because B-PE is manufactured from a microorganism, it was found to vary in purity and composition from lot to lot. In addition, B-PE is highly photosensitive which is a severe drawback when fluorescence intensity decay is used in the assay in that B-PE requires special handling.
Another problem with the prior art ORAC assay is that since only one standard is used, calculating the antioxidant capacity of the sample based on the fluorescence intensity decay of the probe in both the sample and the standard incorrectly assumes that a direct ratio between the antioxidant capacity of the standard and the sample could be made. This, however, is not true.
Still another problem with the prior art ORAC assay is that percloric acid was added to biological samples to separate proteins from the sample. The inventors hereof discovered that percloric acid, itself a strong oxidizing agent, yielded false low antioxidant capacity readings.
Finally, the prior art ORAC procedure involved a long dwell time of up to 75 minutes.